DNA TOPOISOMERASES--DNA INTERACTIONS AND AT CONTROL

我研究的长期目标是了解 线粒体生物发生的详细机制 酶的结构和功能以及线粒体DNA水平 组织和表达。 该提案的具体目标是 开始阐明 DNA 拓扑异构酶在 线粒体DNA的复制和代谢。 特别是， ATP 与 I 型核和线粒体的相互作用 将研究拓扑异构酶。 已经表明， 不依赖于 ATP 的 I 型拓扑异构酶实际上受到以下因素的抑制 ATP。 由于这种效应已在人类拓扑斯中观察到 白血病、HeLA 和小牛胸腺细胞，我们假设这 ATP 对拓扑异构酶 I 活性的调节可能是一种普遍现象 哺乳动物细胞。 核苷酸酶的详细信息 相互作用以及该调节的机制用于 将阐明体内和体外研究。 这些酶在线粒体 DNA 复制中的作用 新陈代谢将通过研究相互作用来定义 拓扑异构酶 I 和 II 与体内线粒体基因组 体外。 这些研究将利用几种抗肿瘤药物 已被证明可以特异性干扰拓扑异构酶 催化链断裂和重新连接过程，使得 蛋白质变性剂的存在，DNA 裂解结果 拓扑异构酶共价连接到 DNA 末端 片段。 特别是，喜树碱将用于探测 topo I-DNA 相互作用和 4'- (9-吖啶基胺)-甲烷 磺酰甲氧基苯胺 (mAMSA) 和表鬼臼毒素 VP-16 VM-26将用于证明topo II-DNA相互作用。 这些 研究应阐明参与的一些细节 topo I 和 topo II 在复制和表达中的作用 线粒体基因组。 拓扑异构酶活性的调节具有普遍意义 但正如提案中详细说明的那样，维持适当的 染色体和染色体外 DNA 超螺旋是 在致癌、诱变和肿瘤发生中起重要作用 因此，这些研究与癌症病因学问题相关 和治疗。

The long-term objectives of my research are to understand the detailed mechanisms of mitochondrial biogenesis at the level of enzyme structure and function as well as at the level of mtDNA organization and expression. The specific goal of this proposal is to begin to elucidate the roles of DNA topoisomerases in the replication and metabolism of mtDNA. In particular, the interaction of ATP with nuclear and mitochondrial type I topoisomerase will be investigated. It has been shown that the ATP-independent type I topoisomerase is actually inhibited by ATP. Since this effect has been observed with topos from human leukemia, HeLA and calf thymus cells, we hypothesize that this ATP regulation of topo I activity may be a general phenomenon in mammalian cells. The details of the nucleotide-enzyme interaction as well as the mechanism of this regulation using in vivo and in vitro studies will be elucidated. The role of these enzymes in mitochondrial DNA replication and metabolism will be defined by investigating the interactions of topoisomerase I and II with the mitochondrial genome in vivo and in vitro. These studies will utilize several antitumor drugs which have been shown to specifically interfere with the topoisomerase- catalyzed strand breaking and rejoining process such that, in the presence of a protein denaturant, DNA cleavage results with topoisomerase covalently attached to the end of the DNA fragment. In particular, camptothecin will be used to probe for topo I-DNA interactions, and 4'- (9-acridinylamin)-methane sulfon-m-anisidide (mAMSA) and the epipodophyllotoxins VP-16 and VM-26 will be used to prove topo II-DNA interactions. These studies should elucidate some of the details of the involvement of topo I and topo II in replication and expression of the mitochondrial genome. Regulation of topoisomerase activity is of general significance but as detailed in the proposal, the maintenance of proper chromosomal and extrachromosomal DNA superhelicity is important in carcinogenesis, mutagenesis and tumorigenesis and as such these studies are relevant to problems in cancer etiology and therapy.